The succulent leaves of certain plants, including tobacco, spinach, soybeans, wheat, cotton and alfalfa, are composed of 10-20% solids, the balance being water. For its parts, the solid portion is composed of a water soluble portion and a water insoluble portion, the latter being made up, for the most part, of the fibrous structural material of the leaf.
The water soluble compounds are divisible into two groups. One group includes compounds of relatively lower molecular weight such as sugars, vitamins, amino acids and other compounds whose molecular weight do not exceed about 10,000. The second group is almost exclusively proteins whose molecular weights are about 30,000 or greater.
The proteins can be resolved into two fractions. One fraction contains a mixture of proteins whose molecular weights range from about 30,000 to 100,000. These proteins are sometimes referred to as "Fraction 2 proteins." The remaining fraction comprises a single protein having a molecular weight of about 550,000 and is sometimes referred to as "Fraction 1 protein."
Fraction 1 protein was first identified in 1947. Subsequent research led to the discovery that this protein was an enzyme involved in photosynthesis. Since then it has been given a number of names. Among these are ribulose 1,5-diphosphate carboxylase, carboxydismutase, ribulose 1,5-bisphosphate carboxylase and ribulose 1,5-di(or bis) phosphate carboxylase-oxygenase.
Fraction 1 protein may compose up to 25% of the total protein content of a leaf and up to 10% of the solid matter in the leaf. In 1970 it was discovered that crystalline Fraction 1 protein could be obtained from tobacco leaves.
Fraction 1 protein, when pure, is odorless, tasteless and colorless and has high nutritional value. In view of these properties, and because it can be obtained in high purity, Fraction 1 protein is considered to have a potentially valuable application as a food supplement for animals and humans. In the case of humans, the additive could be a component of high protein or other special diets. It has, for example, been suggested as a supplement to the diet of persons who require dialysis because of kidney disease.
Despite its relative abundance in cultivated plants, Fraction 1 protein is not a commercially important product since the processes known to the art for obtaining it from vegetable matter are not commercially feasible.
Three basic processes for isolating Fraction 1 protein have been described in the published literature. Each published method begins with pulping the leaves, or leaves and stalk of the plant, followed by expressing a green juice from the pulp. The green juice, which contains finely particulate green pigmented material, is clarified for example, by filtration or centrifugation, to separate the finely particulate solid matter from the liquid. The resulting liquid is brown in color.
The first method described for isolating Fraction 1 protein involved concentration of Fraction 1 protein simultaneously with its partial separation from lower molecular weight compounds in the brown juice by molecular filtration. Using a molecular sieve whose pores would pass smaller molecules without passing Fraction 1 protein, the brown juice was placed under pressure so that small molecules would pass through the pores.
The solution containing the Fraction 1 protein was concentrated about ten-fold and then dialyzed to remove additional small molecules in the solution. Dialysis was accomplished using a collodion type dialysis bag. The pores of the bag would not permit passage of the Fraction 1 protein but allowed the smaller molecules to escape through the bag into water. During dialysis, crystals of Fraction 1 protein formed.
The second method developed to isolate the Fraction 1 protein involved passing the brown juice obtained from the leaves through a Sephadex chromatographic column. Sephadex consists of water insoluble microscopic beads of polymerized sugar. Either Sephadex G-25 or G-50 was used to perform the separation. Selection of proper beads permits small molecules to penetrate to the interior of their structure to the exclusion of larger molecules. The larger molecules, therefore, are only found in the liquid in the interstices between the tightly packed Sephadex beads. This interstitial space is referred to as the "void volume".
To achieve effective separation, the volume of brown juice cannot exceed about 25% of the total volume of the beads. The beads are first equilibrated with a buffer and a volume of brown juice, containing the same buffer, is then layered on top of the Sephadex column.
The brown liquid is eluted from the column using the buffer solution. As the juice moves down the column, the passage of small molecules is retarded since they penetrate the interior of the beads. The large Fraction 1 molecules, on the other hand, move at a faster rate down the column through the labyrinth formed by the interstices between the beads and emerges from the column as a clear brown solution. However, elution results in at least two-fold dilution of the solution. Removal of the smaller molecules changes the environment around the molecules of Fraction 1 protein which leads to crystallization.
The most recently described method provides passage of the brown juice through a Sephadex G-25 column as described above. If Fraction 1 protein does not crystallize, as is the case with the extract of all plants except tobacco, ammonium sulfate is added until the solution is 30-50% saturated. This leads to precipitation of an amorphous material which is collected by centrifugation. After separation, the precipitate is redissolved in a smaller volume of buffer than that from which it was precipitatd to which is added 8% polyethylene glycol. This mixture is placed in an open dish adjacent to another open dish containing silica gel and the two dishes confined in a closed vessel. Water is gradually evaporated from the protein solution and absorbed by the silica gel. With the passage of time, crystals of Fraction 1 protein develop.
It will be clear to those skilled in the art that the prior art processes described above are either time consuming, expensive or both. However, in our copending application Ser. No. 78,505, we describe a simple process for isolating Fraction I protein comprising the steps of converting the leaves to a pulp, heating the liquid portion of the pulp to a temperature below that which causes the protein to denature followed by cooling the liquid portion to a temperature at which Fraction 1 protein, i.e., ribulose 1,5-diphosphate carboxylase crystallizes.